1. Field of Use
These teachings relate generally to a system and method for microscopy illumination in general and more particularly to an adjustable TIRFM illumination apparatus.
2. Description of Prior Art
Various mechanisms are often employed in fluorescence microscopy applications to restrict the excitation and detection of fluorophores to a thin region of the specimen. Elimination of background fluorescence from outside the focal plane can dramatically improve the signal-to-noise ratio, and consequently, the spatial resolution of the features or events of interest. Total internal reflection fluorescence microscopy (TIRFM) exploits the unique properties of an induced evanescent wave or field in a limited specimen region immediately adjacent to the interface between two media having different refractive indices. In practice, the most commonly utilized interface in the application of TIRFM is the contact area between a specimen and a glass cover-slip or tissue culture container. A collimated light beam propagating through one medium and reaching, such an interface is either refracted as it enters the second medium, or reflected at the interface, depending upon the incident angle and the difference in refractive indices of the two media. Total internal reflection is only possible in situations in which the propagating light encounters a boundary to a medium of lower refractive index. Its refractive behavior is governed by the well known Snell's Law.
Although light no longer passes into the second medium when it is incident at angles greater than the critical angle, the reflected light generates a highly restricted electromagnetic field adjacent to the interface, in the lower-index medium. This evanescent field is identical in frequency to the incident light, and because it decays exponentially in intensity with distance from the interface, the field extends at most a few hundred nanometers into the specimen in the z direction (normal to the interface).
In a typical experimental setup, fluorophores located in the vicinity of the glass-liquid or plastic-liquid surface can be excited by the evanescent field, provided they have potential electronic transitions at energies within or very near the wavelength bandwidth of the illuminating beam. Because of the exponential falloff of evanescent field intensity, the excitation of fluorophores is restricted to a region that is typically less than 100 nanometers in thickness. By comparison, this optical section thickness is approximately one-tenth that produced by confocal fluorescence microscopy techniques. Because excitation of fluorophores in the bulk of the specimen is avoided, confining the secondary fluorescence emission to a very thin region, a much higher signal-to-noise ratio is achieved compared to conventional wide field epifluorescence illumination. This enhanced signal level makes it possible to detect single-molecule fluorescence by the TIRFM method.
Generally, two types of TIRF illumination are known in the prior art. The first prior art illumination is by means of a prism. The fluorescence is collected through an objective and is formed at a charge-coupled-device (CCD) camera. It is understood that the TIRF illumination is performed on the side pointing away from the objective. This has the disadvantage that the specimen to be studied has to be prepared on the prism, because the evanescent lighting field is excited at the boundary surface between the prism and the specimen. This type of preparation is expensive. In contrast thereto, specimens are prepared as a rule on a thin cover glass. The sample is generally prepared on a glass surface coupled to the prism using a coupling medium of glycerol, or oil. This is an inconvenient method and difficult to set up and align. It typically restricts the sample from Brightfield imaging.
In the second type of TIRF illumination disclosed, for example in FIG. 9 of WO 20061127692 A2, the specimen can be prepared by a standard procedure on a cover glass because here the TIRF illumination is performed through the microscope objective.
Typically, however, this arrangement has had the disadvantage that the microscope objective has to possess a high numerical aperture in order to make it possible to have a large angle of incidence necessary for high resolution for the excitation light T. As a result, there are increased demands upon the glasses used whereby the number of glass types available is reduced. For example, immersion media and front lenses with a correspondingly higher index of refraction have to be used. In addition, the number of lenses for image correction has to be increased, as a rule, so that manufacturing expense rises and transmission decreases. If the specimen for the TIRF excitation is illuminated with different light wavelengths, so must the angle of incidence, in order to guarantee a high resolution, for all the wavelengths to be identical, the complexity of the microscope and with it its manufacturing expense increase further.
Although there were disadvantages to through the lens TIRF the challenges stated are generally well addressed in current objective lens design. While through the lens TIRF is not as pure as Prism type TIRF due to internal reflections and auto fluorescence within the objective lens assembly, in practice they perform extremely well.
However, commercial solutions to implement these new lenses into microscopy systems have been thus far complex and expensive; using a light path which is either common or redundant to an EPI illumination light path.
For example, referring to FIG. 1 there is shown a schematic diagram illustrating prior art conventional TIRF combined with Far field fluorescence. The prior art configuration shown in FIG. 1 includes objective lens 92, dichromatic assembly 94, prism 910, camera 912, a conventional TIRF assembly 930, and an EPI Lamp assembly 79.
The dichromatic assembly 94 comprises fixed filters 95,96 and dichromatic mirror 97. The simplified representation of the conventional TIRF assembly 930 includes lenses 89a, 89b, and 89c. Also shown is a laser source 89d. Similarly the EPI Lamp assembly 79 includes lenses 79a and 79b. The assembly also includes a light source 79c and reflector 79d. 
Still referring to FIG. 1 it can be seen that the emitted light paths for the camera 912, the TIRF assembly 930, represented by 91c, 91d and 91e, 91f, respectively are redundant to the EPI light paths generated by the EPI Lamp assembly 79 (not shown for clarity).
Thus, it will be readily appreciated that prior art solutions are complex, as well as expensive. In order to have both TIRF and Far field fluorescence capability, the hardware associated with each capability needs be stacked, one over the other. This adds redundancy to the optical path and about 3 inches to the height of a microscope.
Therefore, there exists a need for a robust, but less complex, adjustable TIRFM illuminator apparatus